The Roles Of Environmental Enrichment In Functional Recovery From Stroke And Its Underlying Epigenetic Mechanisms

Ischemic stroke is a several result of brain tissue necrosis caused by occlusion of cerebral blood vessels.About 60%-80%of acute stroke are caused by cerebral infraction worldwide.Recent years have witnessed significant advance in hyper-acute stroke care which significantly reduced stroke-death rate.However,stroke sequelae,including cognitive and motor dysfunction,seriously affect life quality of stroke patients worldwide and remain a huge economic burden to humane society.How to promote functional recovery from stroke becomes one of the urgent challenges faced by therapists and researchers.Tissue plamnipen activator(tPA)is the only treatment for hyper-acute stroke approved by Food and Drug Administration(FDA).However,only a small percentage of stroke patients benefit from tPA treatment because of its narrow window for thrombolysis,its neurotoxicity and increasing risk of intracerebral hemorrhage.Currently,there is still a lack of clinical treatment to promote functional recovery after stroke.It is reported that the brain region near stroke damage,namely the peri-infarct zone,plays critical roles in rehabilitation,as it shows heightened neuroplasticity.Delayed physical rehabilitation such as task-specific behaviour training,constraint induced movement therapy(CIMT)and environemantal stimuli can promote stroke recovery.It is well documented that environmental enrichment(EE)can enhance adult brain neuroplasticity and alter cognitive and motor outcome after Alzheimer disease and stroke.But the molecular and cellular mechanisms underlying this phenomenon remain poorly understood.Therefore,we designed two parts of experiment:(1)to investigate whether EE promotes functional recovery from photothrombotic stroke and the cellular and molecular mechanisms underlying EE-induced adult neuroplasticity after stroke.(2)to investigate epigenetic mechanisms underlying EE and stroke recovery.In order to study the role of EE in stroke recovery,we firstly tested the photothrombotic stroke model,and the behavioral experiment confirmed its reliability.The results of foot faults in the grid-walking task and asymmetry index in the cylinder task indicated that motor deficits were clearly presented the first week after stroke induction,then they improved in the following several weeks,but still remained at a high injury level four weeks after stroke.Standard environment(SE)-housed mice were housed in a standard size cage(3-4 mice/cage).The EE mice were housed in a spacious cage(90*70*30)(6-8 mice/cage),containing various colorful and texture toys,tunnels,shelters and running wheels for voluntary exercise.The EE mice were transfered into EE five days after photothrombotic stroke.Grid-walking task and cylinder task were performed at 4 d,12 d,19 d,26 d and 33 d after stroke to assess motor function.Our results suggested that housing in EE for one week is sufficient to improve stroke recovery significantly and persistently.To better understand how EE promotes stroke recovery,we measured neuroplasticity-related gene expression,including synapsin,spinophilin and post-synaptic density protein 95(PSD-95).We found that EE reversed stroke-induced downregulation of these proteins.Next,we tested whether EE enhanced structural neuroplasticity in the peri-infarct cortex after stroke.Mice were housed in EE for one week,and Golgi-Cox staining was conducted at 43 days after stroke.We found that EE significantly increased spine density,number of dendrites,length as well as branching of dendrites.BDA staining results indicated that EE remarkably promoted axonal sproutig after stroke.We also detected the expression of GABA transporter1(GAT1),GABA transporter3/4(GAT3/4).The results of western blotting suggested that EE increased the expression of GAT1.Accordingly,we performed chromatin immunoprecipitation(ChIP)to study whether the upregulation of these proteins was via epigenetic mechanisms.we found that the genes upregulated by EE also showed higher acetylation in their promoter regions.Next,we performed whole-cell voltage-clamp recordings in in vitro brain slices.We found that EE reversed the ischemia-induced increase in Itonic.Collectively,our results suggested that EE improved functional recovery from stroke via enhancing neuroplasticity of peri-infarct cortex after stroke.To study the roles of HDAC2 in EE-induced functional recovery from stroke,we generated cortex specific Hdac2 conditional knock-out(CKO)mice by crossing HDAC2~flox/floxlox/flox mice with Emx1-Cre transgenic mice.We found that EE was ineffective in HDAC2 CKO mice.Moreover,We microinjected adeno virus vector Ad-HDAC2-Flag and its control Ad-inactive-HDAC2-Flag into the peri-infarct cortex of mice immediately after photothrombotic stroke.Western blotting indicated this treatment significant increased HDAC2 in the peri-infarct cortex.We found that overexpression of HDAC2 remarkably impaired EE-induced functional recovery after stroke.Moreover,we generated adeno-associated virus vector AAV-CAG-EGFP-Cre and its control AAV-CAG-EGFP.We injected them into the peri-infarct cortex of HDAC2~flox/floxlox/flox mice immediately after photothrombotic stroke.This treatment substantially downregulated HDAC2 expression.We found EE did not alleviate moter deficits in AAV-CAG-EGFP-Cre treated group.Together,these results suggested that EE-induced functional recovery from stroke was HDAC2activity-dependent.Moreover,we found EE induced cortical acetylation histones 4(H4)and increaed HDAC2 activity as soon as three days after treatment.These data indicated that EE promoted functional recovery from stroke via HDAC2-mediated epigenetical mechanisms.In conclusion,these results indicated that EE improved stroke recovery via increasing HDAC2 activity and increasing neuroplasticity-related genes expression.Meanwhile,upregulation of HDAC2 activity loosened histone structure and increased BDNF expression,which lead to upregulation of GAT1 expression and decrease in tonic inhibition.